The invention relates to methods for the determination of vitamin B6 in samples as well as to reagent compositions for assaying a sample for vitamin B6 and to a test kit suitable for carrying out the methods according to the present invention. Further the invention relates to the use of such methods for the application to different analyzing devices such as microtiter plate readers and fully automated clinical chemistry analyzers (autoanalyzers).
Vitamin B6 is a water-soluble vitamin that exists in three major chemical forms: pyridoxine, pyridoxal and pyridoxamine. It performs a wide variety of functions in the human body and is essential to maintain a good state of health. For example, vitamin B6 is needed as a co-factor for more than 100 enzymes involved in protein metabolism. It is also essential for red blood cell metabolism, the nervous and immune systems need vitamin B6 to function efficiently and it is also needed for the conversion of the amino acid, tryptophan, to niacin (another vitamin). The body also needs vitamin B6 to generate haemoglobin, where vitamin B6 helps to increase the amount of oxygen bound to haemoglobin. In general, vitamins are important for the immune system because they promote the growth of white blood cells which directly fight against infections. In addition, it helps to maintain the health of lymphoid organs (such as thymus, spleen and so lymph nodes).
Vitamin B6 also helps to maintain blood glucose (sugar) within a normal range. When caloric intake is low, the body needs vitamin B6 to help to convert stored carbohydrates or other nutrients to glucose to maintain normal blood sugar levels. A shortage of vitamin B6 will limit these functions.
According to Leklem (Modern Nutrition in Health and Disease, 9th ed., Baltimore: Williams and Wilkins, 1999: 413-421) vitamin B6 is needed for the synthesis of neural transmitters such as serotonin and dopamine. Bernstein (Ann. NY Acad. Sci. 1990; 585:250-60) discovered that these neural transmitters are required for normal nerve cell communication. It is also described that there is a relationship between vitamin B6 concentrations and a wide variety of neurologic disorders such as seizures, chronic pain, depression, headache and Parkinson's disease.
Vitamin B6 was also recommended to treat carpal tunnel syndrome (Copeland and Stoukides, Ann Pharmacother 1994; 28:1042-4). It is still advised to take 100 to 200 milligrams of vitamin B6 per day in cases of carpal tunnel syndrome.
Vitamin B6 has also become a popular remedy for treating the discomforts associated with premenstrual syndrome (PMS).
Vitamin B6 is a water-soluble compound that was discovered in the 1930s during nutrition studies on rats. The vitamin was named pyridoxine to indicate its structural homology to pyridine. Later it was shown that vitamin B6 could exist in two other slightly different chemical forms, termed pyridoxal and pyridoxamine. All three forms of vitamin B6 are precursors of the biologically active compound known as pyridoxal-5′-phosphate (PLP).
PLP acts as a coenzyme in all transamination reactions, and in some decarboxylation and deamination reactions of amino acids. The aldehyde group of PLP forms a Schiff-base linkage with the s-amino group of a specific lysine group of the aminotransferase enzyme. The α-amino group of the amino acid substrate displaces the s-amino of the lysine residue in the active site of the enzymes. The resulting aldimine is deprotonated to become a quinoid intermediate, which in turn accepts a proton at a different position on the molecule to become a ketimine. The resulting ketimine is hydrolysed so that the amino group remains on the complex.
PLP is also active in the condensation reaction towards heme synthesis.
Such versatility arises from the ability of PLP to covalently bind the substrate, and then to act as an electrophilic catalyst, thereby stabilizing types of carbanionic reaction intermediates. Overall, the Enzyme Commission (www.chem.qmul.ac.uk/iubmb/enzyme) has catalogued more than 140 PLP-dependent activities.
Vitamin B6 is found in a wide variety of foods including fortified cereals, beans, meat, poultry, fish and some vegetables.
Clinical signs of vitamin B6 deficiency are rare in young people of industrialized nations. Many older individuals, however, have low blood levels of vitamin B6 which may suggest a marginal or sub-optimal vitamin B6 nutritional status. Vitamin B6 deficiency can occur in individuals with poor quality diets that are deficient in many nutrients. Symptoms occur during later stages of deficiency, when intake has been very low for an extended time. Signs of vitamin B6 deficiency include dermatitis (skin inflammation), glossitis (a sore tongue), depression, confusion and convulsions. Vitamin B6 deficiency can also cause anaemia. Some of these symptoms can also result from a variety of other medical conditions different from vitamin B6 deficiency. Therefore, it is important to have a physician evaluating these symptoms, including the determination of the Vitamin B6 status, so that appropriate medical care can be given (Institute of Medicine, National Academy Press, Washington, D.C., 1998).
Individuals with a poor quality diet or an inadequate vitamin B6 intake over an extended period may benefit from taking a vitamin B6 supplement, if they are unable to increase their dietary intake of vitamin B6. Alcoholics and older adults often show low vitamin B6 concentrations because of the limited variation of their diet. Alcohol also promotes the destruction and loss of vitamin B6 from the body.
Asthmatic children treated with the drug, theophylline, may need to take a vitamin B6 supplement (Weir et al, Ann. Allergy 1990; 65:59-62). Theophylline decreases vitamin B6 levels and theophylline-induced seizures have been linked to low body stores of the vitamin.
Classical syndromes for vitamin B6 deficiency are also seborrheic dermatitis-like eruption, atrophic glossitis with eruption, atrophic glossitis with ulceration, angular cheilitis, conjunctivitis, intertrigo and neurologic symptoms of somnolence, confusion and neuropathy.
Vitamin B6 is also a co-factor for glutamic acid decarboxylase, an enzyme that converts glutamate to GABA. Therefore, the concurrent increase of the excitatory neurotransmitter, glutamate, and the decrease of the inhibitory neurotransmitter, GABA, resulting from vitamin B6 deficiency potentially manifesting in seizures.
The term vitamin B6 includes several related molecules of which the active entity is pyridoxal-5′-phosphate (PLP). PLP serves as a coenzyme for many enzymes, primarily transferases, lyases and isomerases (Percudani R, Peracchi A. EMBO Rep 2003; 4(9):850-4). This predominantly prokaryotic cofactor is essential for eukaryotes for basic cell metabolism. Well known is the requirement of PLP as a coenzyme for cystathione-β-synthase (CBS), catalyzing the important step of the conversion of homocysteine to cysteine. A deficiency of PLP or vitamin B6 leads to increased levels of CBS substrates, including elevated homocysteine. Higher homocysteine levels correlate with higher risk for cardiovascular diseases, in particular an elevated risk for heart attack (Schwammenthal Y, Tanne D. Lancet Neurol 2004; 3(8):493-5). In a prominent study low blood PLP concentrations have been shown to be an independent risk factor for coronary heart disease (Folsom A R, et al. Circulation 1998; 98:204-10).
An overdose of pyridoxine can cause a temporary loss of certain nerves such as the proprioceptory nerves; causing a feeling of disembodiment, common with the loss of proprioception. This condition is reversible when supplementation is stopped.
Although vitamin B6 is a water-soluble vitamin and is excreted in the urine, very high doses of pyridoxine over long periods of time may result in painful neurological symptoms known as sensory neuropathy. Symptoms include pain and numbness of the extremities, and in severe cases difficulty in walking. Sensory neuropathy typically develops at doses of pyridoxine in excess of 1,000 mg per day. However, there have been a few case reports of individuals who developed sensory neuropathies at doses of less than 500 milligrams daily over a period of months. In order to prevent sensory neuropathy in virtually all individuals, the Food and Nutrition Board of the Institute of Medicine of the Unites States of Amerika, Wash., set the tolerable upper intake level (UL) for pyridoxine at 100 milligrams per day for adults. Because placebo-controlled studies have generally failed to show therapeutic benefits of high doses of pyridoxine, there are only a few indications to exceed the UL of 100 milligrams per day. Studies have shown that in the case of individuals diagnosed with autism, high doses of vitamin B6 given with magnesium have been extremely beneficial.
Measurement of the concentration of vitamin B6 is therefore an important diagnostic tool. It has been carried out in the past by several different testing methods such as biologically deductive, biochemical and physico-chemical methods.
Methods using High Pressure Liquid Chromatography (HPLC) analysis such as developed by Torres-Sequeiros et al (Chromatografia 2001; 53:S236-9) and Argoudelis C. J. (Chromatografia 1997; 790:83-91) do have the advantage that the determination of vitamin B6 is rapidly and practicably realizable, however, the detection limit often does not exceed 6 micromols/liter which makes them unsuitable for the determination of physiological concentrations. Moreover, the equipment needed is costly and their maintenance can be time-consuming. The hand-ling of HPLC analyzers requires well trained and qualified technicians.
In this context, there is also an example referred to a method using the commercially available ClinRep® Komplettkit “Vitamin B6 in Plasma/Vollblut” (RECIPE CHEMICALS+INSTRUMENTS GmbH) whereas the sample (e.g. plasma or blood) is first treated with a protein precipitation reagent followed by HPLC analysis of the supernatant, which also makes this method costly and involves the handling by highly qualified personel. Further, a similar test-kit suitable for HPLC analysis is available from Chromsystems GmbH, Germany (www.chromsystems.com).
Further, the U.S. Pat. No. 6,426,194 B1 discloses a method for quantification of PLP in biological samples. This method comprises the reaction of samples with the apoenzyme form of a PLP-requiring-enzyme which can generate a product when PLP is present, preferably one that is determinable by colorimetry or fluorescence. Apoenzymes as used in the U.S. Pat. No. 6,426,194 B1 are homocysteine/methionine alpha-gamma lyases, which have been depleted of PLP usually associated with them. This method does not involve the disadvantages regarding costs and specifically trained staff as compared to methods requiring HPLC, but it may lead to poor and imprecise results, particularly in the pathologically low range and the lower normal range below approximately 30 to 50 nanomols/liter.
Another well established method known within the state of the art has been developed by the applicant (www.buhlmannlabs.ch; product “RK-VB6”). This method relates to radio-enzymatic determination of the vitamin B6 concentration in a sample. In this method, 3H-tyrosine is decarboxylated by the vitamin B6 dependent enzyme, tyrosine apodecarboxylase (Y-apoDC) from Streptococcus faecalis, to 3H-tyramine. The activity of tyrosine apodecarboxylase is quantitatively dependent on the amount of pyridoxal-5′-phosphate (PLP) present in the reaction mixture. The 3H-tyramine thus produced is selectively extracted into the scintillation cocktail (the excess of 3H-tyrosine remains in the aqueous phase) and can be measured by liquid scintillation counting. This method is very sensitive (down to 2 nanomols/liter) and reproducible in the determination of Vitamin B6 in a wide variety of sample materials.
A further example method was reported by Gregory et al (J. Nutr. 1991; 121:177-86). This method was carried out by using deuterium-labelled pyridoxine or pyridoxine-β-glucoside.
However, the use of radioactive compounds in the latter two methods also has many disadvantages. Extensive safety precautions such as use of lead shielding and special waste treatment procedures must be undertaken in their storage, use and disposal. Expensive equipment is needed for radioactive counting. The radioactive decay of the isotopes used does not only reduce the amount of radioactivity available for detection over time, but may also initiate chemical reactions that damage the remaining reagents reducing sensitivity further. Thus, the storage of suitable reagents is limited to several months. In addition, these methods are not automatable.
Further, Mass Spectroscopy (MS) has been used for the determination of vitamin B6. It has, however, been found that up to date such methods are not suitable for practice-oriented serial measurements due to imprecise quantification (Borsch, Dissertation [PhD Thesis]: “Optimierte HPLC-Analytik zur Bestimmung der Bioverfügbarkeit von freiem and gebundenem Vitamin B6 in physiologischen Konzentrationen beim Menschen”, Giessen 2002).
As a result of the disadvantages of the methods already known within the art, there is a need for other determination methods of vitamin B6, which allow a very precise determination but show less disadvantages associated with the methods already known. Further, such a method should be rapid and easily practicable, without the need of too costly equipment and highly qualified staff. Finally there is a need for a method which is also suitable for a commercially available kit with sufficient component stability.